The dual nature of Entamoeba histolytica, as currently defined, has been further examined. One of the implications of the proposed conversion of NP amebae to the P form is that stresses encountered during the axenization process trigger the conversion. Several attempts to axenize NP isolates by methods which routinely produce axenic cultures of P isolates all failed. Axenization of a single NP isolate was finally achieved using a novel axenic culture system. Most important, this NP isolate after several months of axenic cultivation has not converted to a P as determined by isoenzyme and riboprinting analyses. Moreover, it differs physiologically from axenized P isolates. This difference, along with the many genetic differences, supports the concept that the two forms are distinct species, E. histolytica Schaudinn 1903 and E. dispar Brumpt 1925. That E. histolytica lacks mitochondria is accepted dogma. However, recently published phylogenetic trees imply that Entamoeba is descended from mitochondria-bearing ancestors. A search in E. histolytica has revealed the presence of a gene that in other eukaryotes encodes a mitochondrial protein. This finding has important implications for our understanding of the metabolism of the parasite. Electrophoretic isoenzyme analysis plays an increasing role in distinguishing between the several species of amebae infecting the human gut. Limitations associated with the use of starch gels, which is the present standard, have been overcome through use of agarose gels. Proteose Peptone #3 can substitute for casein peptones, which are routinely used in media for axenic culture of Entamoeba.